ABSTRACT Polycystic ovary syndrome (PCOS) affects 5-7% of reproductive-age women, and is characterized by increased androgen levels, anovulation, and infertility. The goal of the proposed studies is to elucidate the mechanism(s) by which DENND1A, a strong PCOS candidate gene identified in genome wide association studies (GWAS) predisposes women to hyperandrogenism and the reproductive consequences of PCOS. DENND1A is a clathrin-binding protein that sits in coated pits at the crossroads of receptor/ligand interactions with downstream intracellular signaling pathways. We have shown that a truncated isoform of DENND1A (called DENN1A.V2, or V2) is increased in PCOS theca cells. Forced expression of V2 in normal theca cells, increases androgen biosynthesis, and expression of key steroidogenic enzymes involved in androgen biosynthesis, converting the cells to a PCOS phenotype. While knock-down of V2 in PCOS theca cells reduces both elevated androgen biosynthesis and steroidogenic enzyme expression resulting in a normal phenotype. We have identified a human DENND1A.V2 monoclonal antibody (V2 hmAB) that inhibits androgen biosynthesis in PCOS theca cells, and transforms the intracellular-signaling/kinome cascade of PCOS theca cells to that observed in normal theca cells. We created a human V2 transgenic (V2-Tg) mouse that has the PCOS phenotype of both augmented androgen biosynthesis and ovarian CYP17A1 gene expression. This proposal will use our PCOS theca cell model systems and the first genetic PCOS mouse model (V2-Tg) to investigate the mechanisms underlying V2 overexpression in PCOS, and the role V2 plays in regulating the genes involved in the PCOS phenotype. We postulate that V2 determines the intensity of LH signaling, as well as the location of signaling by routing receptor/ligand containing vesicles to different pathways thus engaging alternate kinase pathways. In Aim 1, we will define the mechanism(s) by which V2 induces a PCOS phenotype, and determine whether V2 acts at multiple cellular loci, including the cell surface, cytoplasmic endosomal vesicles, and in the nucleus to selectively regulate genes involved in androgen excess. The role of DENND1A.V2 will be probed with our V2 hmAB. Kinome-profiling studies will identify alterations in key signaling cascades in normal and PCOS theca cells associated with V2. The role of V2 in the transcription of genes encoding androgenic enzymes and global gene expression will also be evaluated. Aim 2 will explore whether epigenetic factors (DNA methylation, microRNAs ) are involved in V2 overexpression in PCOS. Aim 3 will utilize our V2-Tg mouse to determine whether altering V2 alone is sufficient to produce a PCOS phenocopy. We will characterize the extent to which expression of V2 in mice promotes the reproductive, steroidogenic, and metabolic phenotypes associated with PCOS in women, and evaluate the how changes in weight impact these phenotypes. We will also determine whether V2 hmAb ?neutralizes? effects of V2 expression. The proposed studies will clarify the molecular pathology of PCOS, and identify novel diagnostic and treatment methods.